The overall goal of this proposal is to gain a better understanding of the central regulation of the hypothalamic-pituitary-thyroid axis (HPT axis). Thyroid stimulating hormone (TSH) is a heterodimeric protein, which is synthesized and secreted from the anterior pituitary, and is essential for activating thyroid hormone (TH) synthesis in the thyroid gland. In response to TSH stimulation, the thyroid generates two thyroid hormones: T4 and T3. T4 is thought to be a prohormone, which is activated by its conversion to a higher affinity ligand known as T3. Systemically derived or locally produced T3 then binds to thyroid hormone receptors (TRs) in the thyrotropin-releasing hormone (TRH) neuron of the hypothalamus or pituitary thyrotroph and inhibits gene expression. Negative control of the HPT axis by TH bound to TRs is generally accepted to be the dominant influence on the axis. In this model, TRH and TSH production are maximal when TH is absent. Our recent studies in KO mice challenge this model. We demonstrate that both TRH and TR-beta are necessary for activation of TSH subunit gene expression and TSH production, even in the hypothyroid state. However, the function of TR-alpha in this process and the mechanism of action of unliganded TR-beta are still unknown. In this proposal, therefore, we will define the role and mechanism of TRH and unliganded TRs in stimulating TSH subunit gene expression in the hypothyroid state. When TH is present, the TR-beta isoforms (principally the TR-beta 2 isoform) mediate TH negative feedback at the level of the hypothalamus and pituitary based on the findings of modestly elevated TSH and TH levels in TR-beta KO mice. In contrast, TR-alpha KO mice actually display slightly reduced TH levels associated with slightly elevated TSH levels. These findings suggest that TR-alpha has no role in negative TH regulation of the HPT axis in the euthyroid state. However, it must play some role in negative regulation because supraphysiologic T3 administration to TR-beta KO mice suppresses TSH synthesis, and most impressively KO of both TR-alpha and TR-beta isoforms markedly elevates both TSH and TH levels in mice. What explains this TR isoform difference? In this proposal, we will test the hypothesis that the lower affinity of TR-alphaiDor T3 explains why it has a limited role in regulating the central axis in the euthyroid state.